Recombinant cytotoxin as well as a method of producing it

ABSTRACT

The subject of the present invention is a method of modifying proteinaceous toxins through the addition of an NLS motif. The resulting cytotoxin facilitates the selective elimination of proliferating cells, particularly tumour cells.

The subject of the present invention is a method of modifyingproteinaceous toxins through the addition of an NLS motif. The resultingcytotoxins facilitate the selective elimination of proliferating cells,particularly tumour cells.

A poison is an organic or inorganic substance which, even at lowconcentrations, has a deleterious effect on living organisms. Poisonsare divided into two basic categories. The first consists of naturalpoisons, produced mainly by pathogenic bacteria, poisonous fungi andplants, as well as venomous animals. The second group of poisonsconsists of anthropogenic poisons. Bacterial toxins (venoms) are variouschemical compounds produced by bacteria which poison a higher organism.They act specifically on various systems (i.e. on the gastrointestinaltract) or cells of an organism (i.e. neurotoxins). These aredifferentiated into exotoxins and endotoxins. Exotoxins, secretedoutside of the live cell are strong venoms and induce specific diseasesymptoms. They have a proteinaceous structure (metabolic product) andthus are sensitive to high temperatures (>60° C.) as well as beingdegraded by digestive enzymes (with the exception of botulinum toxin andStaphylococcus enterotoxins). They have strong antygenic properties, andanatoxins made therefrom are used to immunise humans and animals. Theyare made mainly by Gram-positive bacteria. These are some of thestrongest toxic substances known. Endotoxins are released only followingthe degradation of the bacterial cell. They are weak venoms, and thesymptoms they induce are not specific. Chemically, these areglycolipopolypeptide complexes (lipopolysaccharide) which most oftenoccur in Gram-negative bacteria in one of the three cell-wall layers.They are poorly antygenic. They are not degraded by digestive enzymesbut are thermostable. Exotoxins secreted by bacteria (but also byplants, fungi and some animals) exhibit cytotoxic properties against ahost cells, usually due to the enzymatic inhibition of proteinsynthesis. The essential condition toxin activity is their binding ofsurface receptors on the target cell and their internalisation throughendocytsis, and then translocation from the endoplasmatic reticulum intothe cytosol. Bacterial exotoxins are currently produced using geneticengineering methods or chemically conjugated with ligands and antibodiesso as to bind to specific cell types. This facilitates the selectivedestruction of disease-altered cell lines. The use of bacterialexotoxins specific for tumour cells is one of the targeted therapeuticstrategies against cancer. Exotoxins secreted naturally by thedisease-causing bacteria Pseudomonas aeruginosa and Diphtheriaetyphimurium are compounds of very high cytotoxicity, sometimes manytimes higher than of classic antitumour drugs. In many cases a singletoxin molecule is capable of killing a cell, which makes them some ofthe most lethal compounds. An exotoxin frequently used to constructfusion proteins with antitumour activity is exotoxin PE from Pseudomonasaeruginosa (Pseudomonas exotoxin, PE) [1]. A molecule of native PE toxinconsists of a catalytic domain connected with a domain that binds areceptor through a central translocation domain, which facilitates thetransfer of the C-terminal catalytic domain into the cytosol (drawing1). So far, the role of the Ib domain of PE remains unknown, but it isknown that it contains a disulphide bridge necessary for moleculematuration. Drawing 2 shows the mechanism of PE intoxication. Due to theinteraction with the host cell, PE binds to the α2 macroglobulins. Priorto entering the cell, the toxin is cleaved proteolytically. Acaroxypeptidase cuts off the terminal lysine which exposes the REDLmotif. Next, the exotoxins are internalised through endocytosisdependent on the receptor. After entering endocytotic vesicles, thetoxin is cleaved proteolytically by furin inside the translocationdomain, and the disulphide bridges hold the formed fragments until theyare reduced. The PE migration pathway in the cell is through the Golgiapparatus and encompasses retention in the endoplasmatic reticulum dueto the REDL signal at the C-terminus of the protein. Then, the freedcatalytic domain is translocated through the reticulum wall into thecytosol. There, the active protein catalyses ADP-ribosylation of His atposition 699 of the translation factor eEF2 and thereby inhibits proteinsynthesis, thereby quickly leading to cell death [1]. The use of PE intargeted therapy entails the replacement of the receptor-binding domainwith an antibody or a portion thereof, a cytokine or growth factor(hence the name immunotoxins). The most frequently used form of PE is afractional exotoxin of 38 kDa composed of amino-acids 253-364 and381-613. Chimeric immunotoxins based on PE molecules are most oftendirected against receptors IL2 and IL6 as well as growth factor TGFα(Tab. 1) [2]. The table below lists information regarding the use ofpropharmaceuticals containing immunotoxins in the treatment of tumours(clinical trials).

TABLE 1 Immunotoxins based on PE in clinical trials, 2009 data [2]Immunotoxin Construction Target antigen Tumors References CD19-ETA′ scFvfused to PE38KDEL CD19 Lymphoma, leukemia Schwemmlein et al. (2007)Anti-Tac(Fv)-PE38KDEL scFv fused to PE38KDEL CD25 CD25 positive tumorcells Kreitman et al. (1994) [LMB2] Anti-Tac(Fv)-PE40KDEL scFv fused toPE40KDEL CD25 Chronic lymphocytic leukemia Kreitman et al. (1992)RTF5(scFv)-ETA′ scFv fused to PE40 CD25 Lymphoma Barth et al. (1998)RFB(dsFv)-PE38 [B1.22] dsFvfused to PE38 CD22 B-cell leukemia Kreitmanet al. (2000a) G28-5 sFv-PE40 scFv fused to PE40 CD40 Burkilt's lymphomaFrancisco et al. (1997) K14(scFv)-ETA′ scFv fused to PE40 CD30 Hodkin'slymphoma Klimka et al. (1999) CD7-ETA scFv fused to PE40 CD7 T-lineageacute lymphoblastic Peipp et al. (2002) leukemia OVB3-PE mAb linked viadisulfide bond Ovary Ovarian Willingham et al. (1987) to PE B3-Lys-PE38[LMB-1] mAb chemically linked to PE38 LeY Various Pastan (1997)B1(dsFv)-PE38 dsFv fused to PE38 LeY LeY positive tumor cells Benhar etal. (1995) B3(dsFv)-PE38 dsFv fused to PE38 LeY LeY positive tumor cellsBenhar et al. (1995) BR96sFv-PE40 [SGN-10] scFv fused to PE40 LeY LeYpositive tumor cells Friedman et al. (1993) IL4(38-37)PE38KDEL IL4 fusedto PE38KDEL IL4-R Breast, SCCHN, pancreas, Leland et al. (2000);Kawakami [NBI-3001] medulloblastoma et al. (2000, 2002); Strome et al.(2002); Joshi et al. (2002) IL13-PE38QQR IL13 fused to PE38QQR IL13-RHead and neck Kawukumi et al. (2001) scFv(FRP5)-ETA scFv fused to PE40erbB2 Ovarian, prostate Wels et al., (1992); Schmidt et al. (2001); Wanget al. (2001) AR209 [e23(Fv)PE38KDEL] scFv fused to PE38KDEL erbB-2Lung, prostate Skrepnik et al. (1996, 1999); Erb-38 dsFv fused to PE38erbB2 Epidermoid carcinoma, breast Reiter and Pastan (1996) MR1(Fv)-PE38scFv fused to PE38 EGFRvIII Glioblastoma Beers et al. (2000) TP38 TGF-αfused to PE38 EGFR Glioma Sampson et al. (2003) TP40 TGF-α fused to PE40EGFR Glioma, prostate, epidermoid Sarosdy et al., (1993); Pai et al.(1991a); Kunwar et al. (1993) 425.3PE mAb chemically linked to PE EGFRBreast Anderssan et al. (2004) A5-PE40 scFv fused to PE40 PSMA ProstateWolf et al. (2006, 2008) SS1(dsFv)PE38 [SSIP] dsFv fused to PE38Mesothelin Ovarian, cervical Hussan et al. (2002) scFv(MUC1)-ETA scFvfused to PE40 MUC1 Breast Singh et al. (2007) 9.2.27-PE mAb chemicallylinked to PE HMW-MAA Gliomblastoma Hjartland et al. (2004)TP-3(scFv)-PE38 scFv fused to PE38 Osteosarcoma Osteosarcoma Onda et al.(2001) antigen TP-3(dsFv)-PE38 dsFv fused to PE38 OsteosarcomaOsteosarcoma Onda et al. (2001) antigen 8H9(dsFv)-PE38 dsFv fused toPE38 Cell surface Breast, osteosarcoma, Onda et al. (2004) glycoproteinneuroblastoma 4D6MOCB-ETA scFv fused to PE40KDEL Ep-CAM Lung, colon, SCCDi Panlo et al. (2003) HB21(Fv)-PE40 scFv fused to PE40 TfR ColonShinohara et al. (2000)

Denileukin diftitox (ONTAK) is at present the only available therapeuticwhich is an immunotoxin. Registered in 1999, it is used in the therapyof CTCL, Cutaneous T-Celi Lymphoma. The FDA report of 16.10.2008 givesit a full marketing permits.

The distribution of cell surface antigens used in targeted therapy isvery often not limited to tumour cells, but is only characterised byincreased frequency in comparison to normal cells. This often causesside effects during the use of the drugs in the form of the destructionof healthy cells, even in tissues and organs with different functions.For example, in the therapy of breast cancer targeted against HER2receptors, one observes the non-specific ingress of immunotoxins intohepatocytes or macrophages, which induces liver damage, and the releaseof cytokines by the macrophages causes subsequent non-specific changes.Newest generation immunotoxins are characterised by a higherspecificity, stemming from the fact that their binding-activity requiresnot one, but two or more factors specific to tumour cells.

The goal of the present invention is to deliver a compound, whoseactivity will be dependent on the phase of the cell cycle and will bepreferably apparent in intensively proliferating cells, particularlytumour cells. It is desirable that the sought substance, in addition tobinding specifically defined epitopes, is subject to specific activationin cancerous cells. This type of substance should be fit for use in theproduction of novel pharmaceutical compositions characterised byincreased therapeutic efficiency w the treatment of tumours as well as alower number of undesirable side effects.

Unexpectedly, the above stated goal has been achieved in the presentinvention.

The subject of the present invention is a method of modifying a proteintoxin through the addition of an NLS motif, which unexpectedly decreasesthe toxicity of the resulting toxin towards non-proliferating cells. Inthe example embodiment of the present invention we design a fusionprotein containing the amino-acid sequence encompassing the sequence ofbacterial exotoxin as well as the sequence of a human NLS motif.

For the purposes of this description, “protein toxins” should beunderstood as natural polypeptides with toxic properties, such as:

-   -   neurotoxins, which hinder neurotransmission,    -   enterotoxins, which damage the gastrointestinal mucosa,    -   cytotoxins, which destroy cells

Protein toxins may be of various origins. Known are the followingtoxins:

-   -   animal i.e.: Cubozoa venom contains protein toxins with        neurotoxic and cardiotoxic properties, which also cause tissue        necrosis; Taipoxin (Oxyuranus scutellatus), inhibits        acetylcholine release from terminal neurons and some cholinergic        neurons of the autonomous nervous system; o-latrotoxin        (Latrodectus) binds with membrane neurexins and causes the        sudden depletion of synaptic vesicles;    -   fungal i.e.: α-amanitin (deathcap mushroom), binds with RNA        polymerase II, at higher concentrations also with RNA polymerase        III, preventing RNA elongation during synthesis; α-sarcin        (Aspergillus giganteus) inhibits protein synthesis by        hydrolyzing phosphodiester bonds in 28S RNA in the large        ribosomal subunit;    -   plant i.e.: Holotoxins (also called class II        ribosome-inactivating proteins) i.e.: ricin (castor oil plant),        abrin (rosary pea), lectin (mistletoe), modecin (Adenia        digitata); Hemitoxins (also called class I ribosome-inactivating        proteins) i.e.: PAP (pokeweed antiviral protein), saporin        (Saponaria officinalis), bouganin (Bougainvillea spectabilis)        and gelonin (Gelonium Multiflorum) [3]; (Holotoxins are composed        of a binding domain and a catalytic domain whereas hemitoxins        contain only a catalytic domain. Plant toxins inhibit the        binding of the elongation factors EF-1 and EF-2 with the        ribosomal 60S subunit by removing the A residue in position 4324        of 28SRNA. Ricin also removes the neighbouring G residue at        position 4323 [4]. The result of such toxin activity is cell        death via apoptosis. Only the enzymatic domain is translocated        into the cytoplasm, and thus the binding domain of holotoxins is        cleaved off through the reduction of the disulphide bond        [5-7].);    -   bacterial i.e.: Neurotoxins: botulin (Clostridium botulinum)        (the activity botulinum toxin is based on permanent affixation        to the neuromotor plate and disruption of muscle contraction.        This is done by the fragmentation of the SNAP-25 protein        essential to acetylcholine secretion from the presynaptic        terminus), tetanus toxin, tetanospasmin (Clostridium tetani)        (tetanospasmin binds to peripheral motor neurons, enters the        axon and from there transfers to neurons of the brain stem and        spinal chord. It then migrates through the synapse to the        presynaptic terminus where it blocks the release of        neurotransmitters (glycine and GABA); Enterotoxins:        streptolysine O (Streptococcus pyogenes), listeriolysine O        (Listeria monocytogenes), alpha-toxin (Staphylococcus aureus)        (these toxins are capable of integrating into the cell membrane        in which they form channels. In this way the porous membrane can        no longer function, and ions begin to egress the cell whereas        water begins to flow inside and the cell may swell and lyse.);        Cytotoxins: collagenases, hyaluronidases or phospholipases are        enzymes which respectively degrade collagen (facilitating deep        penetration of tissue) and membrane phospholipids; Shiga toxin,        Stx (Shigella dysenteriae) this protein is composed of 6        subunits: 5 B subunits, responsible for binding the toxin to its        receptor—globotriaosylceramide (Gb3) of a eukaryotic cell and an        A subunit, which is proteolysed following endocytosis to        peptides A1 and A2. StxA2 is an enzyme which cleaves an adenine        off 28S ribosomal RNA. This inhibits tprotein synthesis in a        cell and its death; cholera toxin (Vibrio cholerae) catalyses        the binding of ADP-ribose to a G-protein subunit which lose its        GTPase activity. It fails to dissociate from adenylate cyclase,        of which it is an activator. Surplus synthesis of cyclic AMP        causes an increased concentration of electrolytes in the        intestinal lumen (storage of chlorides and inhibited potassium        absorption), which causes constant water flow into the        intestines; dyphtherotoxin (Corynebacterium diphtheriae), a        transferase which transfers ADP-ribose from NAD+ to eEF-2        (ADP-ribosylation) and in this way inhibits the translocation        and thus the elongation of a polypeptide chain; exotoxin A        (Pseudomonas).

For the purposes of this description, “immunotoxins” should beunderstood as complexes of antibodies or their fragments with toxins,chemically bound. The antibody is directed against structures on thetumour cell surface. Most often, recombinant immunotoxins produced by E.coli are used, such as:

-   -   human interleukin-2 (IL-2) combined with dophthitoxin        (denileukin diftitox)—reacts with the IL-2 receptor. This drug        is registered for the treatment of dermal T-cell lymphomas. It        has also been tested in CLL patients resistant to other        antileukaemia drugs [8]. Denileukin diftitox is administered at        a rate of 18 μg/kg/day in a 60 minute infusion over five days at        21 day intervals. Up to 8 combined cycles have been used. In 12        patients, reduced leukaemic cells have been observed in the        blood of over 80% of the patients, and in 6 a decrease in lymph        node volume. 6 of 22 patients who received at least 2 cycles        fulfilled the criteria for full or partial remission.    -   BL22—a recombinant immunotoxins containing an IgG immunoglobulin        fragment, which recognizes antigen CD22, conjugated with the        exotoxin of Pseudomonas [9]. The antibody is highly active in        the case of hairy cell leukaemia [10.11]. A high efficacy was        also observed against CLL but not against CR [11,12]. Currently,        a BL22 mutant termed HA22 is undergoing clinical trials [9].

For the purposes of this description the human “NLS” motif (nuclearlocalization signal or sequence) should be understood as an amino-acidsequence motif warranting intracellular transport of a protein into thenucleus. It comprises a sequence of positively charged amino-acids,lysines and arginines (so-called single NLS), meeting the consensusK-K/R-X-KR with the sequence: KKKRKR [13].

An example use of the present invention is exotoxin A of Pseudomonasaeruginosa modified such that in the amino-acid sequence it contains anadditional NLS motif: KKKRKR added at position -633, behind proline -632from the amino end (as shown in sequence 1) in relation to the nativeprotein.

The next subject of the present invention are nucleotide sequences ofDNA, cDNA and mRNA encoding exotoxin A of Pseudomonas aeruginsa modifiedsuch that at position 1933 in relation to the native sequence theadditionally contain a motif encoding NLS, taking into account thedegeneration of genetic code, meaning that all DNA encodes the proteinwith the amino-acid sequence according to the present invention as ithas been defined above. Particular embodiment of the nucleotide sequenceaccording to the present invention is sequence 2.

The next subject of the present invention are modified proteins,derivatives of exotoxins containing the protein fragment describedabove, with the modification described above, as well as the DNAsequences encoding them.

The next subject of the present invention are recombinant expressionvectors as well as expression cassettes containing said DNA sequences.

The next subject of the present invention is the production of saidproteins through overexpression in cells and in extracellular systems.

The next subject of the present invention is the use of said proteins totreat eukaryotic cells.

The next subject of the present invention the use of said proteins inthe production of pharmaceutical compositions.

The description of the present invention is illustrated by the attachedfigures. FIG. 1 represents a schematic representation of the structureof the exoPE toxin [14]. FIG. 2 represents the mechanism of PEintoxication.

To better understand the present invention defined above, the presentdescription also contains an example embodiment of the presentinvention. This example, however, should not be treated as limiting thescope encompassed by the present invention. The example embodiments areillustrated by the attached figures, wherein FIG. 1 shows the map of thevector pJ206, wherein the frame encloses the restriction sites EcoRI andHindIII. FIG. 2 shows the map of the vector pUC57, where the frameencloses the restriction sites for EcoRI and HindIII. FIG. 3 in turnshows an X-Ray film with visible signals corresponding to GFP (GFP),native exotoxin (PET) as well as modified exotoxin (PET mod1). FIG. A.shows the effect of the selected chimeric toxins on live humanfibroblasts, 3T3, neutral red method after 24 h, where: GFP—translationfrom the vector with GFP (translation control), PETm1—translation fromthe vector with the modified toxin whereas PET is the native toxin.

EXAMPLE 1

The sequence encoding the modified exotoxin was designed with thefurther addition of elements necessary to obtain (via in vitrosynthesis) proteins containing a tag in the form of 6 histidineresidues. The nucleotide sequence of the entire expression cassette isshown as sequence No. 3 whereas the protein it encodes has theamino-acid sequence termed sequence 4 (in the sequence list).

The expression cassette containing: a promoter for the T7 polymerase, aribosome binding site, a start codon, a linker with the His-tag as wellas a sequence encoding the modified exotoxin; was obtained throughchemical synthesis performed by the GenScript company. This cassette wasthen cloned by the producer into the vector pJ206 between therestriction sites EcoRI and HindIII (FIG. 1). From such a vector wedigested out the entire expression cassette and transcloned it into theplasmid pUC57 (FIG. 2) using the EcoRI and Hind III restrictases. In thesame way we synthesized and prepared the vectors used to expressproteins that were the controls in the experiment: unmodified (native)exotoxin A of Pseudomonas aeruginsa as well as GFP.

The vector containing the insert, the expression cassette for themodified exotoxin as well as vectors for the expression of the nativeexotoxin and GFP were used for in vitro transcription and translationusing the commercial “RTS 100 E. coli HY Kit containing E. coli lysate(5Prime)”. Proteins synthesized in this fashion were purified onNi-NTA-agarose (Qiagen) and dialysed against PBS, and then concentratedusing Amicon centrifuge filters. The concentration of the resultingproteins in subsequent purification steps was estimated using the BCAmethod (Tab. 2).

TABLE 2 Concentrations of proteins obtained via in vitrotranscription/translation, estimated using the BCA method. proteinconcentration [μg/ml] preparation cleaned on preparation followingNi-NTA-agarose concentration on Amicon filters GFP 75 227 PET <10 39PET_mod1 22 64

The molecular mass of the resulting proteins was evaluated usingelectrophoresis on Agilent microchips. The results are shown below (Tab.3) and reflect the predicted mass of the resulting polypeptides.

TABLE 3 Molecular mass of the proteins obtained through in vitrotranscription/translation, evaluated using the Agilent microchip. mass[kDa] GFP 30 PET 74 PET_mod1 76

To confirm that the resulting fusion protein of the desired mass wasobtained, we performed Western blot analysis. Detection was performedusing an antibody against the His-tag, conjugated with HRP (horseradishperoxidase). As is shown in FIG. 3, all resulting proteins bound theanti-His-tag antibody. The resulting signals corresponded to a mass ofabout 30 kDa (GFP) and 75 kDa (native and modified toxins).

We then tested the effect of the modified toxins on the growth ofNIH/3T3 mouse fibroblasts and its selective cytotoxic effect onintensively dividing cells. FIG. 4 shows a compilation of survivabilityresults of the treated cells. In the case of modified exotoxin, weobserved differences dependent on the stage of development of theculture (confluence of 100% and 40%). Intensively dividing cells(initial confluence 40%) were more sensitive to the modified exotoxinthan 100% confluent cells, and the difference was about 11%.

References:

-   1. Kreitman R J. Recombinant immunotoxins containing truncated    bacterial toxins for the treatment of hematologic malignancies.    BioDrugs., 2009, 23(1):1-13.-   2. Wolf P, Elsässer-Beile U. Pseudomonas exotoxin A: from virulence    factor to anti-cancer agent. Int J Med Microbiol. 2009, 299, 161-76.-   3. Bolognesi A, Polito L, Tazzari P L, et al. In vitro anti-tumour    activity of anti-CD80 and anti-CD86 immunotoxins containing type 1    ribosome inactivating proteins. Br J Haematol. 2000; 110: 351- 361.-   4. Endo Y, Mitsui K, Motizuki M, Tsurugi K. The mechanism of action    of ricin and related toxic lectins on eukaryotic ribosomes. J Biol    Chem. 1987; 262: 5908-5912.-   5. Bolognesi A, Tazzari P L, Olivieri F, Polito L, Falini B,    Stirpe F. Induction of apoptosis by ribosome-inactivating proteins    and related immunotoxins. Int J Cancer. 1996; 68: 349-355.-   6. Hughes J N, Lindsay C D, Griffiths G D. Morphology of ricin and    abrin exposed endothelial cells is consistent with apoptotic cell    death. Hum Exp Toxicol. 1996; 15: 443-451.-   7. Bergamaschi G, Perfetti V, Tonon L, et al. Saporin, a ribosome    inactivating protein used to prepare immunotoxins, induces cell    death via apoptosis. Br J Haematol. 1996; 93: 789-794.-   8. Frankel A E, Surendranathan A, Black J H, White A, Ganjoo, Cripe    L D. Phase II clinical studies of denileukin diffitox fusion protein    in patients with previously treated chronic lymphocytic leukemia.    Cancer. 2006; 106: 2158-2164.-   9. Robak T. Novel monoclonal antibodies for the treatment of chronic    lymphocytic leukemia. Curr Cancer Drug Targets. 2008; 8: 156-171.-   10. Kreitman R J, Wilson W H, Bergeron K, et al. Efficacy of the    anti CD22 recombinant immunotoxins BL22 in chemotherapy resistant    hairy cell leukemia. N Engl J Med. 2001; 345: 241-247.-   11. Robak T. New agents in chronic lymphocytic leukemia. Curr Treat    Options Oncol. 2006; 7: 200-212.-   12. Kreitman R J, Squires D R, Stetler-Stevenson M, et al. Phase I    trial of recombinant immunotoxins RFB4(dsFv)-PE38 (BL22) in patients    with B-cell malignancies. J Clin Oncol. 2005; 23: 6719-6729.-   13. Chelsky D, Ralph R, Jonak G. Sequence requirements for synthetic    peptide-mediated translocation to the nucleus. Mol Cell Biol. 1989    Jun; 9(6):2487-92.-   14. Kreitman R J. Immunotoxins for targeted cancer therapy. AAPS J.    2006 Aug 18; 8(3):E532-51.

1. A method of producing a recombinant cytotoxin, the method comprisingadding an NLS motif to the amino-acid sequence of a known protein toxinor its derivative, wherein the result is a recombinant cytotoxin with adecreased overall cytotoxicity in comparison to the cytotoxicity of theknown protein toxin or its derivative.
 2. A fusion protein comprising anamino-acid sequence of a protein toxin and a human NLS motif amino acidsequence.
 3. The fusion protein according to claim 2, wherein theprotein toxin is a known protein toxin from animals, fungi, plants orbacteria.
 4. The fusion protein according to claim 2, wherein theprotein toxin is exotoxin A of Pseudomonas aeruginsa.
 5. The fusionprotein according to claim 2, wherein the protein toxin is animmunotoxin.
 6. The fusion protein according to claim 2, wherein the NLSmotif comprises the amino-acid sequence KKKRKR.
 7. The fusion proteinaccording to claim 2, comprising the amino-acid sequence shown as SEQ IDNO: 1 or SEQ ID NO:
 4. 8. A fusion protein according to claim 2 for usein anti-tumour therapy.
 9. A polynucleotide encoding a fusion proteinaccording to claim
 2. 10. A polynucleotide according to claim 9,comprising the nucleotide sequence shown as SEQ ID NO: 2 or SEQ ID NO:3.
 11. A biologically active vector, comprising a polynucleotideaccording to claim 9.